The three-dimensional structure of the wild type and mutants of homeodomains in the NK-2 class, specifically the vnd/NK-2 homeodomain, and the full length CSX/NKX-2.5, and NKX-3.1 proteins, both in the free state and bound to DNA have been investigated. Ad additional construct that contains both the vnd/NK-2 homeodomain, a linker and the NK-2 specific domain also were studied in the free and DNA bound states. This latter study shows that only the homeodomain itself interacts directly with DNA. Our transgenic studies show that the Y54M alteration acted as a knockout, where ability to down-regulate ind and msh apparently is suppressed completely. The acidic domain deletion mutant showed limited down-regulation capability. By contrast, the tinman and the NK-2 box deletion mutants both behaved fully as functional vnd/NK-2 genes in their ability to repress ind and msh. Another single amino acid residue mutation in the homeodomain, H52R, stabilized the homeodomain both structurally and thermally. Nevertheless, this mutation in a transgenic experiment was toxic to the embryo and completely obviated growth as soon as expression of mRNA was initiated. The NMR determined tertiary structures of the Y54M vnd/NK-2 homeodomain in vitro both free and bound to DNA were compared with the wild type analog. The only structural difference observed for the mutant homeodomain was in the complex with DNA and involved a closer interaction of the methionine with A2 rather that C3 of the DNA. This change in the interaction of the homeodomain with DNA resulted in significant alteration in the lifetimes of the homeodomain-DNA complexes. The lifetimes were studied in four systems; 1) the wild type homeodomain bound to its cognate DNA, 2) the wild type homeodomain bound to a sequence of DNA that contains 5' - CAATGG - 3', which is the consensus sequence of homeodomains that contain methionine in position 54, 3) the Y54M homeodomain bound to wild type core DNA consensus sequence, and 4) the Y54M homeodomain bound to the DNA that contains 5' - CAATGG 3' as its core. The lifetimes of the wild type homeodomain bound to the wild type core DNA consensus sequence (i.e., #1) were at least one order of magnitude longer than possibilities 2 - 4. The implication or hypothesis here is that the lifetime of the transcriptional activation complex must be sufficiently long to insure proper embryonic development. To investigate this hypothesis further, CAT and beta-galactosidease reporter gene assays were carried out using several 750 base-pair sequences of the 5' upstream region of the vnd/NK-2 gene as promotors. Preliminary results demonstrated that alterations of either 5' - CAAGTG - 3' or 5' - CAATGG - 3' DNA fragments significantly altered the expression of both CAT and beta-galactosidease. Both activation and repression was seen depending on the specific changes made. These changes resulting from a single amino acid residue replacement constitute the molecular basis for the phenotypic alterations observed upon ectopic expression of the Y54M vnd/NK-2 gene during embryogenesis. An important relationship between the regulatory role of the NK-2 class of homeobox genes in development and highly explicity homeodomain-DNA interactions is thereby established. These studies also demonstrate the relation between sequential and structural modularity with functional modularity, where in the above example, only the homeodomain appears to be necessary to down-regulate ind and msh. Studies on mutant CSX/NKX-2.5 homeodomains associated with atrial septum defect show that the tertiary structure of the Y54C mutant protein is not altered but that the interaction with the functional DNA binding sites is modified. The Y54C CSX/NKX-2.5 protein is totally non-functional. This modification of the interaction of the protein with the DNA likely constitutes the molecular basis of the disease. Recent structural studies suggest that tthat only part of the CSX/NKX-2.5 protein that is structured in the absence of binding to DNA or other protein factors is the homeodomain itself.